Cryoprecipitation is a long established means for preparing fibrinogen concentrates from human and other mammalian plasma as fibrin sealants in surgical repair. The ubiquitous physiological role of fibrinogen and fibrin in the phenomena and mechanism of coagulation, internal restructuring, wound healing, and tissue repair has been extended over the past scores of years to a concentrate, processed variously from plasma for applied tissue bonding under such descriptive terms as fibrin glue, fibrin adhesive, fibrin weld, fibrin sealant, and so on.
The clinical use of fibrinogen prepared from plasma by various methods of cryoprecipitation and by chemical insolubilization has gradually emerged for such early uses as hemostyptic adhesive powder with small open vessels (Bergel, S., Deutsch Med. Wochenschr. pp. 633-665, 1909), as a hemostatic agent in cerebral surgery (Grey, E. G., Surg. Gyn. Obst., Vol. 21, pp. 425-454, 1915), in suturing peripheral nerves (Matras, H. et al., Wien. Med. Wochenschr., 1972), and gradually expanded to the repair of traumatized tissues (Brands, W. et al., World J. Surg., Vol. 6, pp. 366-368, 1982), and anastomoses or restructuring of cardiovascular, colon, bronchial sections, severed nerve endings, and other anatomical discontinuities currently in wide-spread practice often replacing or augmenting conventional suturing. In such clinical applications, the native fibrinogen content in plasma averages 513 milligrams per decaliter (mg/dcl) according to standard clinical assays, ranging from as low 229 mg/dcl to as high as 742 mg/dcl, based on photometric measurements of turbidity from clotting (Castillo, J. B., et al. Thrombosis Res., Vol. 55, pp. 213-219, 1989). In a typical reference (Dresdale, A. et al., Surgery, Vol. 97, p. 751, (1985); also published PCT patent application WO 86/01814), the stated sequence of cryofreezing, thawing, and centrifuging produces a fibrinogen concentrate of extremely low productivity of only 2.16 percent (2160 mgm/dcl). The resulting fibrinogen concentrates of the prior art are too low in solids content, are further diluted with added thrombin for conversion to fibrin state, and therefore lacking viscous contact tenacity of low viscosity, very much like that of water and appreciably lower than of the initial plasma from which it derived.
In order to correct and improve upon the needed productivity with defined and specified qualifications tests and performance standards, lacking in or unattainable from currently available methods, a novel and more efficient and accountable process was devised as described in the applications Ser. No. 07/562,839, now abandoned, hereinafter '839, and Ser. No. 07/855,752, now abandoned, hereinafter '752. In the '839 application, the principal objective was to attain a higher level Of solids content in fibrinogen concentrates by applying controlled thermal drift throughout the integrated cryoprecipitation, thawing, and centrifuging steps. This process resulted in an unexpected increased productivity, and enhanced adhesion and bonding, as demonstrated by in vivo animal tissue adhesion bonding, wound healing, and restored biomechanical tissue integrity. None of the prior art provides such essential descriptive details on process productivity, process efficiency, and product qualifications with supporting tests for a broadened range of the cryoprecipitated native proteins supplemented with such macromolecular structures as polysaccharides, glycoproteins, and the like for biomedical applications in surgical tissue reconstruction.
In support of the inventions described in this and the '752 and '839 applications, cryoprecipitated plasma product qualification tests have been devised to serve as a basis for specifications and uniform performance standards for regulatory compliance in direct clinical applications, for large scale production from pooled plasma, and for autologous small scale single lot preparations of enhanced plasma protein concentrates. The process of the present invention, which prepares fibrinogen concentrates, is especially important in view of the prevalent risk of viral infections, notably numerous forms of hepatitis and human immune deficiency virus (HIV), from pooled or single donor non-autologous sources.
This application extends the thermal drift process further with a novel intervening step wherein the cryoprecipitate is added to a transfer phase containing naturally occurring or synthetic macromolecular and functional entities of relatively low molecular weight. The macromolecular entities are intended to enhance contact adhesion to tissues and to assure safe and effective in vivo tissue bonding, healing, and restored biomechanical integrity. The phase transfer media includes antibiotic, antifibrinolytic, anticoagulant supplements, and preservatives and stabilizers, for extended storage stability shelf life.
Apart from cryoprecipitation, alternate means for separating and concentrating fibrinogen or plasma protein in currently available methods involve chemical or solvent precipitating procedures by admixtures with concentrated salt solutions, such as semi-saturated sodium chloride and saturated ammonium sulfate, and by cold ethanol and other low molecular weight organic compounds, notably amino acids such glycine, and numerous combinations thereof.
However, these chemical precipitating methods impose varying degrees of denaturation (Putnam text, The Plasma Proteins, Academic Press, Section 3,1975) in contrast to non-chemical cryoprecipitation. Chemical precipitations with organic solvents or additives are used mostly for preparing high purity fibrinogen stripped of the natively associated symbiotic plasma proteins, notably the low molecular weight proteins, glycoproteins, and polysaccharides which remain dissolved by virtue of higher degree of solubility. Consequently, it is only possible slowly, at elevated temperatures and only in low concentration, to re-dissolve the chemically precipitated and thusly denatured fibrinogen and plasma proteins from lyophilized form; the solubility of the lyophilized denatured fibrinogen can be increased with specific chemical formulation with decrease in viscosity but still in denatured state. The cryoprecipitation process of the invention distinguishes clearly from chemical precipitation by preserving the solubility of the cryoprecipitates, without the need for any re-dissolution, to as high as 40 percent protein solids content with corresponding increase in viscosity.
In the course of chemical preparations of fibrinogen concentrates, the native, plasma proteins include valuable associated mucopolysaccharides and glycoproteins in their varied acetylated and aminic forms of mucopolysaccharides, discarded according to the practices of the prior art in the course of the chemical preparations of fibrinogen for fibrin sealants. Now with the present and preceding :inventions, it has been discovered that the discarded molecular entities cryoprecipitated from re-cycled supernatants continue to provide concentrates with significantly enhanced viscous adhesion in tissue bonding in actual in vivo tissue and in vivo animal tests as described in the previous application, Ser. No. 07/855,752, now abandoned, and as shown in ensuing examples of this application. The essential processing steps of this invention are discoursed as follows and detailed with a number of Examples typifying the preferred embodiment with different transfer media and different mammalian plasma types supplementations.
Cryoprecipitation
Cryoprecipitation heretofore has not been generally recognized as a preferred method for making enhanced high solids fibrinogen concentrationis with retained associated native mucoproteins and mucopolysaccharides for adhesive viscous quality in preference to chemical fractional precipitation that specifically strip off the solubilized associated mucoproteins and mucopolysaccharides. Such adventitious chemical stripping can be expected to impose major physical conformational changes in the molecular form and shape of native fibrinogen structure (see Putnam text, The Plasma Proteins, Section 3 and Section 4, Academic Press, 1975) commonly referred to as denaturing with marked changes in physical, chemical and biological characteristics.
In the prior art cryoprecipitation starts off with cryofreezing, that is, deep freezing down to about -80.degree. Centigrade with a wide range of temperature-time variables but with no indication of the effect of the varied temperature-time kinetics on productivity or on measured, quantitized product properties or specifications as described and assessed in the preceding application Ser. No. 07/855,752, filed Mar. 23, 1992, now abandoned. Rather the prior art predisposes to infer that longer periods of cryofreezing and thawing are necessary for attaining some undefined merit and with no measures of productivity and product quality of the fibrinogen tissue sealant with or without the numerous and diverse associated native plasma proteins and polysaccharides. For instance, the clinical preparations in the prior art commence with cryofreezing at -80.degree. C., specified for at least 6 hours (Gestring, G. F., et al., Vascular Surgery, p. 295, 1983); later this was increased to at least 18 hours (Dresdale, A., et al., Annals of Thoracic Surgery, Vol. 40, p. 885, 1985); and again later for at least 24 hours (Spotnitz, W. D., et al., American Surgeon, p. 461, 1937). Clearly this teaching to increase the cryofreezing time is misleading. Furthermore these prior art prolonged time schedules are prohibitive for needs in autologous emergency tissue sealing for it was discovered, as described in application Ser. No. 07/562,839, on Aug. 6, 1990, now abandoned, on process engineering for producing fibrinogen concentrates, that the cryofreezing time at -80.degree. C. can be reduced down to 1 hour and less for expedient, emergency surgical needs with specified, substantially increased productivity hitherto unstated in the prior art with regard to yield and solids content of stained fibrinogen concentrate.
In the present invention, an innovative, intervening feature is provided with the direct transfer of the native cryoprecipitate from the plasma following the cryofreezing with continued solubilization into a pre-prepared transfer medium throughout the thawing and centrifugation of tile engineering process system. As described in the original application following cryofreezing during phase transfer, controlled thawing is the next essential and critical process step during which the solid heterogeneous crystalline-like frozen mush is transformed into the two phases. The upper supernatant phase comprises residual icing, also referred to herein as icing, in the form of a glacialized homogeneous solid plug of ice transformed from the cryofrozen mush. This residual icing has not been recognized in the known prior art as a controlling feature for process productivity within the range from about 5 weight percent to about 95 weight percent icing depending upon the applied temperature-time thermal drift schedule through the solidus--liquidus transition temperature.
With prolonged thawing, either as the usual separate process or simultaneously during centrifuging, the ice progressively melts during the thermal drift along with restricted re-dissolving of the low and intermediate molecular weight proteins, glycoproteins, and mucopolysaccharides depending upon the residual icing. The control of the thermal drift from cryofreezing is critical to the quality of the cryoprecipitate concentrate, the solids concentration assay and the distribution of the numerous associated plasma proteins and mucopolysaccharides through the solidus--liquidus equilibrium transition temperature depicted as follows:
______________________________________ Process phases ______________________________________ cryoprecipitation thawing centrifugation -0.degree. C. .fwdarw. &gt;0.degree. C. (solidus) (de-icing) (liquidus) ______________________________________
wherein the solid plasma releases the cryoprecipitated insoluble fibrinogen and its relatively soluble associated proteins and mucopolysaccharides, which are important for enhanced tissues bonding, retained with the cryoprecipitate concentrate by the control of the level of residual icing for attaining the desired solids contents in the concentrates. The ratio of the fibrinogen to the associated plasma components proteins and mucopolysaccharides is thus regulated by the time-controlled thermal drift of the solidus--liquidus transition as the more soluble plasma components re-dissolve with increasing time at the thawing equilibrium temperature. Each component has its own solidus--liquidus transition temperature. Thus, various components may be released and/or retained depending on the selected solidus--liquidus transition temperature.
The associated plasma macromolecular proteins serve as intrinsic bioadhesives, characterized as mucoproteins and chemically known as glycoproteins indigenous with fibrinogen and are intended to be retained as component portions of the various cryoprecipitated compositions of the concentrates. Included in the cryoprecipitated products are numerous hematological factors involved in the clotting mechanism and cell growth factors involved in the healing of the rejoined tissue incisions for which the macromolecular proteins contribute enhanced viscosity for the peremptory handling and dispensing qualifications. All of these ancillary indigenous plasma components, considered valuable and indispensable for firm tissue bonding, are returnable as part of the cryoprecipitate concentrates by restricting their dissolution with the temperature-time thermal drift control of the process through the thawing phase at the transition equilibrium temperature.
Thawing Control
The extent of thawing by this invention is controlled by the de-icing in the supernatant phase by the measure of residual glacialized ice retained in the form of a solid plug, hitherto not recognized in the prior art. The ice plug by reason of its slightly lower density than its ice water phase floats to the top of the supernatant fluid, is readily withdrawn, weighed or measured as thawed weight or volume, and rated in terms of percent residual icing from the initial weight or volume of plasma. The extent of thawing, based on the percent residual icing, is thus regulated by applying appropriate temperature-time schedule either as a separate thawing procedure or preferably simultaneously with the centrifuging time. The overall process time thus provides a measure of process time-efficiency while the productivity is determined as the product of (a) the cryoprecipitate yield in grams multiplied by (b) the percent solids assay converted to and expressed in milligrams; these two items of materials constitute the indispensable means for further assessments of materials balance and process improvements from the important basic solids assay of the initial plasma. These productivity items in turn affect the attained product qualifications of this invention measured principally in terms of measured viscosity and ultimate tissue bonding strengths.
Thus the extent of the retention of the fibrinogen associated plasma components is dependent upon the applied thermal drift time from the cryogenic state through the icing equilibrium with minimal prolongation of time in the liquidus watery phase in the developing supernatant phase during which the native associated mucopoly-saccharides, mucoproteins and hematological factors continue to redissolve from the cryoprecipitated state during prolonged thawing time. In this regard the redissolved associated plasma components can be recovered by recycling the separated, decanted supernatant plasma or serum by repeating the complete sequence of the thermal drift from cryofreezing to thawing, separately or preferably simultaneously with the centrifuging operation.
The thawing temperature-time schedule according to published prior art methods is not specific, ignores the critical temperature-time variables, and in no instances provide correlations to either the productivity in terms of an appropriate materials balance or to product qualifications of the attained plasma cryoprecipitate for effective tissue bonding or tissue sealant applications. For instance, the prior art varies from such indefinite temperature-time kinetics of thawing such as at +4.degree. C. "when liquid" (Gestring, supra); at 4.degree. C. "for several hours" (Dresdale, supra); and at 1.degree. C. to 6.degree. C. for 20 hours (Siedentop, K. H., et al., Laryngoscope, Vol. 95, p. 1075, 1985). In no instance of this typical prior art is there indication of the concentrate productivity in terms of gram yield, percent solids content, and corresponding dry solids content milligrams, as a matter of providing the customary engineering materials accounting or balance for assessing process efficiency with reference to the solids materials contents of the initial plasma from which the cryoprecipitate is derived in a single cycle fraction (such as Fraction I, II, III, etc.) by repeated recycling of the separated supernatants.
In the published cited references, prolonged thawing can only lead to continuing redissolving of the valuable and useful plasma proteins but in no instances is there any indication of residual icing during the temperature-time thermal drift. The references disclose the inordinate loss of valuable fibrinogen and its associated plasma entities notably the proteins and useful mucopolysaccharides with fibrinogen concentrates as low as 2.16 percent content (Dresdale, supra). The need for minimizing the temperature-time thermal drift is made evident by controlling the solidus--liquidus temperature-time to the retention of residual icing within a range from about 5 weight percent to about 95 weight percent. This is to allow maximum thawing at the equilibrium transition temperature on the one hand and for minimal redissolving of the cryoprecipitates on the other hand.
Centrifuging
Following and during thawing, the cryoprecipitate is sedimented further into the transfer phase medium from the thawing plasma by means of centrifugation into the lower pre-prepared viscous phase comprising a solution of natural and synthetic macromolecular polysaccharide of various chemical configurations in such media as sterile water, normal saline, Ringers lactate solution, spent supernatant fluids, and even fractional portions of the plasma. In addition to providing enhanced viscous adhesion and supplementation preservatives, stabilizing agents, antibiotics, and so on, these different phase transfer media, with or without physiological additives, can provide transfer environments either markedly different from, or the same as that of the initial plasma. This in turn can be expected to produce cryoprecipitated concentrates of endless variations and ratios of the myriads of native plasma constituents.
Centrifugation following thawing with the cryoprecipitate phase transfer can be carried out either as a separate process operation, as usually practiced in the prior art, or preferably simultaneously, with thawing at the selected overall temperature-time thermal drift of the processing system. Centrifugation involves a wide range of speed (RPM), gravitational force (xg), and with appropriate combinations of temperature-time schedules that must be rigidly specified especially for product standards intended for surgical or clinical use. The prior art include such variations as, for instance, unspecified cold centrifuging at 2300 xg for 10 to 15 minutes (Gestring, supra); 1000 xg for 15 minutes (Dresdale, supra); 5,000 RPM (unspecified xg) at 1.degree. to 6.degree. C. for 5 minutes (Siedentop, supra); and at 6500 xg for 4 minutes at +4.degree. C. (Spotnitz, supra); in no instances of this prior art is there any indication of productivity or materials balance or appropriate qualification tests for preemptive in vitro tissue adhesion or in vivo animal testing for safe and effective tissue sealant applications.
Given the wide variations of the foregoing temperature-time schedules for each of the three process operations, the known prior art provides no cogent, discernable criteria for providing an efficient process engineering system from a source material as complex as human plasma for producing defined cryoprecipitate products by applying a novel phase transfer from plasma to a pre-prepared receiving substrate for enhanced viscosity and adhesive strength for safe and effective tissue bonding.